Probing conformational changes in Dictyostelium myosin II via cysteine engineering

Myosins couple ATP hydrolysis to the vectorial sliding of nearby actin filaments. To facilitate studying of myosin head conformational changes, we have generated functional mutant Dictyostelium myosin IIs missing exposed cysteine residues. This myosin gene can be further mutated to introduce one or two cysteines at defined locations, in preparation for crosslinking and spectroscopic studies. The “Cyslite” myosin has six out of seven native cysteines in the motor domain changed to other residues, yet retains activity both in vivo and in vitro.;Nucleotide induced conformational changes in the active site pocket may be translated into an angular change in the neck domain through the actions of a relay helix-loop subdomain, which may produce torque on the crankshaft-like proximal domain of the lever arm during the power stroke and subsequent recovery stroke. However, no structural information exists as to whether this contact is maintained in an actin bound state, as would be predicted by this model. To test the model, we introduced cysteines on opposing faces of this interface, and induced disulfide crosslinking. The crosslinked mutant binds actin strongly in the absence of ATP and binds actin weakly in the presence of ATP, much like the wildtype myosin head domain. Thus, these two domains do not need to separate in the weak to strong actin binding transition.;Myosin is thought to generate force through rotation of its “lever arm” neck domain. To measure the extent of lever arm rotation, we used cysteine engineering to facilitate donor dye labelling of a light chain bound to the lever arm domain and acceptor dye labelling at an appropriate location on the catalytic head domain. Steady-state and time-resolved fluorescence resonance energy transfer measurements reported the distance between these two dyes as a function of nucleotide, from which the amount of lever arm domain rotation could be inferred. The results demonstrate that bound Mg.ADP.P i, and not bound Mg.ATP, biases the lever arm towards two distinct prestroke angle states. This FRET-based sensor should be useful for readout of lever arm angle changes in future studies.